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1004 Br J Ophthalmol 2000;84:1004–1007 Topical ophthalmic â blockers may cause release of histamine through cytotoxic eVects on inflammatory cells

Luc M van Beek, Marcel Mulder, Nicolaas J van Haeringen, Aize Kijlstra

Abstract lent antigen (allergen) results, via a molecular Aim—To evaluate the eVects of â blockers cascade, in an increase in the intracellular cal- used in ophthalmology on the release of cium concentration. This in turn leads to histamine from mixed cell preparations release of histamine and other preformed containing human leucocytes and ba- mediators from the granules of the mast cell. sophils. Cross linking of the IgE antibodies also Methods—A mixed leucocyte and ba- activates the enzyme adenyl cyclase which pro- sophil preparation was obtained from motes the synthesis of cyclic adenosine mono- venous blood of healthy non-atopic volun- phosphate (cAMP) from ATP. Cyclic AMP teers. Cell preparations were then incu- inhibits degranulation, thus forming a negative bated with , metipranolol, feedback loop.5 , or . After incubation for Mast cells and basophils express â receptors 1 hour the histamine content of the super- at their surface; these receptors are also natant was analysed by automated fluoro- coupled to adenyl cyclase.6 Activation of â metric analysis. Cell viability was tested receptors increases the intracellular cAMP by measuring lactate dehydrogenase concentration which then acts as a second (LDH) concentrations. messenger. Blocking the â receptors may thus Results—Betaxolol and metipranolol in disturb the negative feedback loop, making the concentrations between 10–2 M and 10–3 M mast cells more susceptible to degranulation. liberated histamine from human blood There have been several reports of a worsening cells in a dose dependent manner. Carte- or recurrence of atopic or anaphylactic reac- olol and timolol had no eVect on histamine tions after starting treatment with systemic â at these concentrations. At the same blockers.7 Department of concentrations LDH was also detected in Betaxolol, carteolol, metipranolol, and Ophthalmology, timolol are blocking agents currently used for Leiden University the supernatants of cell suspensions incu- â Medical Centre, bated with metipranolol or betaxolol. the treatment of . We investigated Netherlands Conclusions—Betaxolol and metipranolol whether these compounds could induce hista- L M van Beek induce substantial histamine release from mine release. As a model we used human human leucocytes, probably as a result of basophils which have similar histamine releas- Netherlands their cytotoxic eVect. ing properties to mast cells.8 Ophthalmic Research ( 2000;84:1004–1007) Institute, Netherlands Br J Ophthalmol L M van Beek Materials and methods N J van Haeringen ISOLATION OF LEUCOCYTES A Kijlstra Topical â blockers are widely used to lower Blood samples from healthy volunteers were intraocular pressure in glaucoma and ocular collected in citrate tubes and suspended in an Department of Allergy, hypertension. Topical â blockers can cause ice cold hypotonic ammonium chloride buVer, CLB and Laboratory serious systemic side eVects1; ocular side causing lysis of erythrocytes. The leucocytes for Experimental and e ects including stinging or burning upon were then washed twice with ice cold HEPES Clinical Immunology, V Academic Medical instillation, foreign body sensation, dry eyes, buVer containing 132 mM NaCl, 6 mM KCl, Centre, University of periorbital dermatitis, conjunctivitis, keratitis, 1 mM CaCl2, 1 mM MgSO4, 1.2 mM 2 Amsterdam, and uveitis have also been described. K2HPO4, 20 mM N-2-hydroxyethylpiperazine- Netherlands Mast cells, which are abundant in the N-2-ethanesulphonic acid (Sigma Chemical M Mulder conjunctiva,3 play a pivotal role in type I hyper- Co, St Louis, MO, USA), 5.5 mM glucose, sensitivity. Mast cells release histamine and and 0.5% (w/v) human serum albumin, pH Department of Ophthalmology other preformed mediators upon stimulation. 7.4, osmolality 280–300 mOsm/l. After wash- A Kijlstra Histamine causes itching, hyperaemia, and ing the leucocytes were suspended in HEPES increases vascular permeability. Earlier reports buVer and incubated with various â blockers. Institute for Animal indicate that histamine is released from iso- The volunteers (n = 14) were free of atopic Science and Health, lated rat mast cells after incubation with disease and were not taking any . Lelystad, Netherlands 4 A Kijlstra certain â blockers. This suggests that some â Several diVerent concentrations (usually three) blockers may have pseudoallergic properties of â blockers were tested in duplicate on each Correspondence to: which may account for some of the ocular side blood sample. All volunteers gave their in- Dr L M van Beek, eVects of these drugs. formed consent and the study conformed to Department of Ophthalmology, Leiden Release of histamine from mast cells (de- the Universal Declaration of Human Rights University Medical Centre, granulation) is the result of either IgE depend- and to the European Convention for the P O Box 9600, 2300 RC ent or IgE independent mechanisms. IgE is Protection of Human Rights and Fundamental Leiden, Netherlands bound to high a nity IgE receptors (Fc RI) Freedoms. [email protected] Y ã on the membrane of mast cells and basophils. The number of leucocytes per aliquot Accepted 26 April 2000 Cross linking of these IgE antibodies by a diva- ranged from 46.2 × 105 to 73.5 × 105. Approxi-

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Release of histamine by topical ophthalmic â blockers 1005

mately 0.5% of leucocytes are basophils,910 Table 1 Histamine release from human leucocyte giving a number of approximately 23.1–36.7 × suspensions of healthy volunteers (n=14) after 1 hour 3 incubation with â blockers (0.01 M) used in treatment of 10 basophils per aliquot. The leucocytes were glaucoma divided into aliquots of 250 µl to which 50 µl of a â blocker solution or phosphate buVered â blocker Mean (SD) histamine release (%) saline (control) was added (final volume Betaxolol 88.5 (5.1) 300 µl). The aliquots were then incubated for 1 Metipranolol 78.8 (6.9) hour at 37ºC. After incubation the leucocytes Timolol 18.5 (11.5) Carteolol 0 (0) were centrifuged (500 g) at room temperature and the supernatant was analysed for lactate dehydrogenase (LDH) or histamine. Directly after starting the reaction, extinc- In some experiments the viability of the cells tion was measured with a photospectrometer was tested using trypan blue staining. Also, in (iEMS Reader MF, Labsystems, Helsinki, Fin- some experiments cell pellets were resus- land) at 340 nm every minute for 10 minutes pended after incubation and the cells were to follow clearance of NADH from the reaction counted again. For the staining procedure cells mixture. It was assumed that the change in the were diluted 1:10 in trypan blue solution 0.3% level of absorption was proportional to the in phosphate buVered saline. Cells were amount of LDH activity in the samples. The counted using phase contrast microscopy amount of LDH was expressed as a percentage (Leica 090-131.001). of the total, corrected for spontaneous release which never exceeded 10%. Total LDH was obtained by sonification of one aliquot of HISTAMINE ASSAY leucocytes. Sonification took place with a Perchloric acid to a final concentration of 2% 30 kHz microprobe (Soniprep 150, MSE, was added to 250 µl of the supernatant of Loughborough, UK) for 15 seconds and was incubated cell suspensions. The supernatants repeated seven times during which the suspen- were then stored at 4ºC until analysis (maxi- sion was kept on ice. After sonification the sus- mum 60 days). The histamine content was pension was centrifuged at 500 for 15 determined at the department of allergy at the g minutes. Spontaneous release was determined central laboratory of blood transfusion (Am- by measuring the LDH content of the control sterdam) by an automated spectrofluorometric supernatant. o-phthalaldehyde assay.11 12 Briefly, this method consists of extracting histamine and coupling it to o-phthalaldehyde. The fluores- AGENTS cence of this complex is then measured by a Betaxolol, carteolol, metipranolol, and timolol spectrofluorometer using an excitation wave- were provided by Tramedico BV, Weesp, length of 355 nm and an emission wavelength Netherlands. They were dissolved in phosphate of 450 nm. It is assumed that the measured buVered saline without preservatives. fluorescence was proportional to the concen- tration of histamine. Results Because the number of basophils and the Initially we tested four â blockers (carteolol, amount of histamine vary between individuals, betaxolol, metipranolol, and timolol) for their the histamine content was expressed as a ability to release histamine from isolated leuco- percentage of the total histamine. Total hista- cytes. These â blockers are currently in use for mine was obtained by adding perchloric acid the treatment of glaucoma. After incubation (final concentration 2 mg/ml) to one aliquot of for 1 hour at a concentration of 0.01 M, betax- leucocytes in each blood sample to cause lysis olol and metipranolol released 79% and 88%, of the cells. This aliquot was then centrifuged respectively, of the total histamine content and histamine was determined using the from the basophils. At the same concentration method described above. Percentages were timolol released only 18% of the histamine and corrected for spontaneous release which was carteolol did not release any (Table 1). defined as the histamine content of the We then constructed dose-response curves supernatant of the control sample. The results for betaxolol, metipranolol, and timolol which were discarded if spontaneous release ex- are shown in Figure 1. Metipranolol and ceeded 15%. betaxolol in concentrations between 10–2 M and 10–3 M liberated histamine in a dose LACTATE DEHYDROGENASE (LDH) ASSAY dependent manner. Timolol liberated signifi- Directly after the incubation experiments the cantly less histamine than either of the other â supernatant of incubated cell suspensions was blockers at the highest concentration (10–2 M). analysed for LDH content. Supernatant (5 µl) To explain these results we hypothesised that was mixed with 200 µl substrate buVer in a 96 the histamine releasing eVect could be either well flat bottom plate. The substrate buVer specific (that is, mediated by the â receptor),

contained 3.8 mM Na2PO4, 1.9 mM KH2PO4, non-specific, or cytotoxic. Trypan blue cell 7.9 µg/ml pyruvic acid, and 15.9 µg/ml counts performed both before and after NADH, pH 7.0. In this mixture LDH converts incubation indicated that cell numbers de- NADH to NAD at a rate determined by the creased during incubation with â blockers amount of LDH present.13 Experiments were (data not shown). To quantitate this eVect, performed at room temperature. Each 96 well LDH was measured in the supernatant after plate contained total, control, and tested â incubation. Results of these experiments are blocker concentrations in duplicate. shown in Fig 2. Again, timolol released signifi-

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1006 van Beek, Mulder, van Haeringen, et al

23 100 Table 2 Lipid solubility of the tested â blockers Betaxolol â 80 Metipranolol blocker Lipid solubility (log P) Timolol Betaxolol 2.81 60 Metipranolol 2.28 Timolol 1.16 p < 0.001 Carteolol 0.82 40

20 fluid. Also, the contact time of the drug will be shorter than the 1 hour used in our experi- Histamine (% of total) 0 ments. Nosal et al15 investigated histamine release 0 0.001 0.01 from isolated rat mast cells after incubation Concentration (M) with metipranolol and several other â blockers. Metipranolol in a concentration of 10–3 M Figure 1 Histamine release from human leucocyte suspensions after incubation for 1 hour at 37ºC with resulted in a 5% histamine release which is in various â blockers. Release is expressed as mean percentage accordance with our results. They did not test with standard deviation of total histamine (n = 14). higher concentrations nor did they test for cell p<0.001 for diVerence between betaxolol and timolol and between metipranolol and timolol at 10–2 M (Student’s t viability after incubation. They further found test). that a â blocker with a higher lipophilicity () induced histamine release at 10–3 M 100 Betaxolol which they concluded was a non-specific eVect Metipranolol caused by perturbation of the cell membrane. 80 16 Timolol Takahashi tested the cytotoxicity of timolol on conjunctival cell cultures and found that 60 timolol 0.5% (10–2 M) did not posses any cyto- toxicity. Similarly, we did not find significant 40 p < 0.001 cytotoxicity with timolol (10–2 M) on leuco-

LDH (% of total) 20 cytes. In our experiments metipranolol and betax- 0 olol had a significantly higher cytotoxicity than 0 0.001 0.01 timolol. Timolol has a considerably lower lipid Concentration (M) solubility than betaxolol and metipranolol 17 Figure 2 Lactate dehydrogenase (LDH) expressed as (Table 2). We feel that, because of their higher percentage of total LDH in supernatant of leucocyte lipid solubility, betaxolol and metipranolol dis- suspensions after incubation for 1 hour at 37ºC with turb the cell membrane integrity and that this various â blockers. Mean values with standard deviations are shown (n = 14). p<0.001 for diVerence between accounts for their higher cytotoxicity. How- betaxolol and timolol and between metipranolol and timolol ever, it should be kept in mind that eye drops at 10–2 M (Student’s t test). need to have a certain lipid solubility to penetrate the cornea.18 In a single drop study cantly less LDH than either betaxolol or meti- timolol was found to reduce exercise tachycar- pranolol. dia in contrast to metipranolol. The authors explained this by the higher lipid solubility of metipranolol permitting rapid cornea penetra- Discussion tion and leaving less metipranolol available for This study shows that histamine is liberated systemic absorption.19 from human basophils by betaxolol and In general, patients find timolol eye drops metipranolol and, to a significantly lesser more comfortable than metipranolol or betax- extent, by timolol. The fact that we detected olol eye drops.20–23 Because the vehicles of the LDH in the supernatant indicated that the â three eye drops are comparable, the low blocker concentrations we used were cytotoxic cytotoxic eVect of timolol may be partly to leucocytes. This cytotoxic eVect may also responsible for its higher degree of comfort. explain the histamine liberation because we do not consider that high concentrations of â This research was supported by a grant of Dr Gerhard Mann blockers are less toxic to basophils than to GmbH, Germany, and Tramedico BV, Netherlands. other leucocytes. Also, in previous studies other authors have used LDH levels to 1 Zimmerman TJ, Baumann JD, Hetherington J Jr. Side eVects of timolol. Surv Ophthalmol 1983;28(Suppl):243– determine the cytotoxic eVects on basophils in 51. similar test conditions.14 2 Akingbehin T, Villada JR. Metipranolol-associated granulo- Betaxolol and timolol formulations are com- matous anterior uveitis. Br J Ophthalmol 1991;75:519–23. 3 Allansmith MR, Korb DR, Greiner JV, et al. Giant papillary mercially available in 0.1%, 0.25%, and 0.5% conjunctivitis in contact lens wearers. Am J Ophthalmol concentrations and metipranolol formulations 1977;83:697–708. 4 Nosal R, Ondrias K, Pecivova J, et al. Histamine liberation are commercially available in 0.1%, 0.3%, and and membrane fluidisation of mast cells exposed to the 0.6% which equals approximately 2.5 × 10–3 to beta-adrenoceptor blocking drug . Agents Ac- tions 1988;23:143–5. 2 × 10–2 M. The cytotoxicity found in these 5 Lichtenstein LM, DeBernardo R. The immediate allergic experiments therefore occurs at concentrations response: in vitro action of cyclic AMP-active and other drugs on the two stages of histamine release. J Immunol that are used in commercial formulations. 1971;107:1131–6. 6 Perper RJ, Sanda M, Lichtenstein LM. The relationship of However, it should be noted that, upon in vitro and in vivo allergic histamine release: inhibition in instillation of an , the concentration primates by cAMP active agents. Int Arch Allergy Appl decreases by dilution with tear fluid and by Immunol 1972;43:837–44. 7 Kaplan AP, Anderson JA, Valentine MD, et al. Beta- binding of the â blocker to proteins in the tear blockers, immunotherapy, and skin testing.

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Topical ophthalmic β blockers may cause release of histamine through cytotoxic effects on inflammatory cells

Luc M van Beek, Marcel Mulder, Nicolaas J van Haeringen, et al.

Br J Ophthalmol 2000 84: 1004-1007 doi: 10.1136/bjo.84.9.1004

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